pH gradients in immobilized amidases and their influence on rates and yields of beta-lactam hydrolysis

The pH gradients developing within immobilized biocatalysts during hydrolys is of penicillin G and glutaryl-7-aminocephalosporanic acid have been estim ated both theoretically and experimentally. For the latter a fluorimetric m ethod for the direct measurement of the average pH value within the carrier during reaction has been developed using the pH-dependent fluorescence int ensity of an enzyme-bound fluorophore determined with a fiber bundle. The t heoretical calculations were based on a model for the hydrolysis with immob ilized enzymes using a kinetic expression with five pH-dependent, measurabl e kinetic and equilibrium constants. The transport reaction differential eq uation which considers the laminar boundary layer has been solved numerical ly for the key component. The calculated values agreed well with the experi mental data. Under the typical reaction conditions of penicillin G hydrolys is the average pH value in the carrier was 1 and 2.5 pH units below the bul k pH (=8) with and without buffer, respectively. The corresponding changes for the hydrolysis of glutaryl-7-aminocephalosporanic acid at bulk pH 8 in the presence of buffer was 0.5. This demonstrates the existence of consider able pH gradients in carriers during hydrolytic reactions, even in buffered systems with negligible mass transfer resistance. The low pH value causes suboptimal reaction rates, reduced equilibrium conversion, and reduced enzy me stability. These pH gradients can be minimised by using buffers with pK values approximately equal to the bulk pH used for the hydrolysis. The pred iction quality of the model has been tested applying it to fixed bed reacto r design. The reduction in rate and yield due to concentration and pH gradi ents can be overcome with simple measures such as high initial pH value and pH adjustments in segmented or recycling fixed bed reactors. Thus, enzymat ic conversions with high yield and high operational effectiveness are achie ved. (C) 1999 John Wiley & Sons, Inc.